Letters in Applied Microbiology ISSN 0266-8254

ORIGINAL ARTICLE

Intraspecies cellular fatty acids heterogeneity of Lactobacillus plantarum strains isolated from fermented foods in Ukraine I. Garmasheva1, O. Vasyliuk1, N. Kovalenko1, A. Ostapchuk2 and L. Oleschenko1 1 Department of Physiology of Industrial Micro-organisms, Zabolotny Institute of Microbiology and Virology National Academy of Science of Ukraine, Kiev, Ukraine 2 Laboratory of Biological Polymer Compounds, Zabolotny Institute of Microbiology and Virology National Academy of Science of Ukraine, Kiev, Ukraine

Significance and Impact of the Study: Cellular fatty acids composition is an important chemotaxonomic characteristic of bacterial cells. At the same time cellular fatty acids play a key role in maintaining the viability of micro-organisms in different environmental conditions. In this study, intraspecies heterogeneity of cellular fatty acids composition of Lactobacillus plantarum strains was examined. This work provides novel and important information about a relationship between cellular fatty acids composition of Lact. plantarum strains and source of isolation or stress resistance profile. Our results showed that cellular fatty acids composition is quite diverse among Lact. plantarum strains derived from different sources and may reflect previous cell’s history. Our findings should be considered in chemotaxonomic studies of lactic acid bacteria and its ecology.

Keywords cellular fatty acid, fatty acids composition, fermented foods, Lactobacillus plantarum, stress resistance. Correspondence Inna Garmasheva, Zabolotny Institute of Microbiology and Virology National Academy of Science of Ukraine, 154 Acad. Zabolotnoho Street, 03143 Kyiv, Ukraine. E-mail: [email protected] 2015/0247: received 4 February 2015, revised 27 April 2015 and accepted 29 May 2015 doi:10.1111/lam.12454

Abstract The intraspecies heterogeneity of cellular fatty acids composition of Lactobacillus plantarum strains isolated from Ukrainian traditional fermented foods was examined. Seven cellular fatty acids were identified. All Lact. plantarum strains investigated contained C16:0 (from 754 to 4983% of total fatty acids), cC18:1 (323–3867% of total fatty acids) and cycC19:0 acids (903-6768% of total fatty acids) as the major fatty acids. The tC18:1 acid made up 147–220% of the total fatty acids. The C14:0 and C16:1 acids were present in small amounts (022–696% and 066–742% respectively) in most Lact. plantarum strains. Differences in relative contents of some fatty acids between Lact. plantarum strains depending on the source isolation were found. Isolates of dairy origin contained slightly greater levels of the C16:0 and tC18:1 fatty acids and lower levels of the cC18:1 than strains obtained from fermented vegetables. The origin of Lact. plantarum strains affects their fatty acids composition, which in turn, appears to be related to their ability to growth under stress factors.

Introduction The species Lactobacillus plantarum is industrially important among lactic acid bacteria (LAB), that is widely isolated from different ecological niches such as fresh and fermented vegetables, meat, fish, dairy products (G omezRuiz et al. 2008; Tanganurat et al. 2009) and intestinal tracts of humans and animals (Bosch et al. 2012). Due to their probiotic properties, some Lact. plantarum strains

had been used for the development of functional and therapeutic foods (Mathara et al. 2008; Turchi et al. 2013). As it was shown by many authors Lact. plantarum is a highly heterogeneous species with a wide range of phenotypic and genotypic properties (Xanthopoulos et al. 2000; Molenaar et al. 2005; Georgieva et al. 2008; Siezen et al. 2010). Lactobacillus plantarum is able to adapt to a variety of habitats which allows this species to spread widely in the environment. It is known that cellular fatty

Letters in Applied Microbiology 61, 283--292 © 2015 The Society for Applied Microbiology

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acids composition play a key role in maintaining the viability of micro-organisms in different environmental conditions (Coulibaly et al. 2009). The numerous studies have described the ability of LAB strains isolated from different sources to grow under stress conditions and synthesize different amount of cellular fatty acids (Guillot et al. 2000; Kimoto-Nira et al. 2009, 2012). At the same time, very little attention is paid to the study of cellular fatty acids composition as the chemotaxonomic species characteristics of lactobacilli (Decallonne et al. 1991; Dykes et al. 1995) and, to our knowledge, no study in which intraspecies heterogeneity of cellular fatty acids composition of Lact. plantarum strains has been reported. It was shown that strains of the same species but of different origin may differ in their properties and ability to adapt to extreme conditions of cultivation (Tanganurat et al. 2009; Siezen et al. 2010), and that the factors such as medium composition, temperature, pH will have a significant influence on the cellular fatty acids composition (Gilarova et al. 1994). In addition, it remains unknown whether the relative content of fatty acids in the bacterial cells is their inherent property under optimal growth conditions or is the result of adaptation to natural econiches. Is it possible on the basis of fatty acids composition to predict the ability of LAB strain to growth under stress conditions? Previously in our laboratory, we characterized some phenotypic properties of Lact. plantarum strains isolated from traditional fermented foods in Ukraine. Significant correlation between the sources of strains and resistance to pH 2, pH 10 and 8% NaCl resistance were found (P < 005). Strains isolated from fermented vegetables were more resistant to these conditions compared with the isolates from dairy products (Vasyliuk et al. 2014b). The purpose of this study was to determine intraspecies diversity of the cellular fatty acid composition of Lact. plantarum strains and assess whether there is a correlation between the ability to grow under stressful conditions and fatty acids profile. In addition, in this study we investigated differences in cellular fatty acids composition between Lact. plantarum strains isolated from fermented vegetables and dairy products of different regions in Ukraine. Results and Discussion To evaluate the intraspecies diversity in cellular fatty acids composition of Lact. plantarum species, we used 109 strains of the Lact. plantarum isolated from different fermented foods in Ukraine and Lact. plantarum type strain CCM 7039T. The best stability of fatty acids composition and the reproducibility of results can be ensured when the strains are resuscitated and cultivated under their 284

optimal conditions (Gilarova et al. 1994). So, in our work the composition of cellular fatty acids of Lact. plantarum strains that were cultured under optimal conditions on MRS medium with Tween-80, which enhances the growth of many LAB (De Man et al. 1960), was studied. Seven cellular fatty acids, namely, tetradecanoic (C14:0), hexadecanoic (C16:0), hexadecenoic (C16:1), octadecanoic (C18:0), octadecenoic (cC18:1 and tC18:1) and cyclopropane (cycC19:0), were identified. All 110 Lact. plantarum strains investigated contained hexadecanoic (from to 754 to 4983% of total fatty acids), cis isomer of octadecenoic (323–3867%) and cyclopropane acids (903–6768%) as the major fatty acids. Trans isomer of octadecenoic acid made up 147–220% of the total fatty acids. The tetradecanoic and hexadecenoic acids were present in small amounts (022–696% and 066– 742% respectively) in most Lact. plantarum strains. To assess the heterogeneity of cellular fatty acids composition of Lact. plantarum strains cluster analyses was used. The tested Lact. plantarum strains were clustered based on their cellular fatty acids profiles (Fig. 1a). As a result four clusters were clearly observed. Strains of clusters I, II and IV were predominantly isolated from dairy products. It should be noted that isolates from soured cream dominated in cluster I, and from cottage cheese – in cluster II. Most of examined Lact. plantarum strains were grouped together with type strain Lact. plantarum CCM 7039T in cluster III and were isolated predominantly from fermented vegetables (Fig. 2). It has generally been reported that Lact. plantarum strains are highly adaptable (Siezen et al. 2010). Thus, despite the prevalence of dairy- or vegetable-derived strains in each cluster according to fatty acids profile, some strains did not group based on their origin. We can assume that this may be a result of the high adaptation capacity of such strains to diverse natural environments. The presence of significant differences in the average fatty acids concentration between strains of each cluster was tested with ANOVA (Table 1). There was no difference of C14:0 content between clusters. Clusters I and IV were characterized by the highest percentage of C16:0, while cluster II – the lowest. Strains of cluster I showed the highest amount of C18:0 acid. Clusters I and III were characterized by the highest content of cC18:1 and the lowest content of tC18:1, while cluster IV was characterized by the highest content of tC18:1 and lowest content of cC18:1. The highest percentage of cycC19:0 were in strains of cluster II, the lowest – in cluster I. Thus, there was a difference between vegetable-derived and dairy-derived strains in terms of fatty acids content. The average percentages of relative fatty acids composition of the Lact. plantarum strains, arranged according to source, are presented in Fig. 3. Isolates of dairy origin

Letters in Applied Microbiology 61, 283--292 © 2015 The Society for Applied Microbiology

Cellular fatty acids heterogeneity of Lact. plantarum strains

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growth in MRS broth

2 952 151 1077 349a 517 790 242a 50 891 1047 682 1010 1043 1081 1123 982 969 1013 1044 923 238a 1011 1121 1128 241 1073 573 200a 94a 191a CCM7039T 560 197a 186 612 1015 188 232 1111 987 199 792 950 321a 1189 47 246 1017 1006 183a 1160 318 922 934 529 743 102 1116 1026 848 977 991 845 1099 1115 212/1a 207a 209a 66 184 1092 830 1094 850 228 345a 998 691 1112 962 198a 794 1120 1045 793 791 795 350a 550 169 193 1056 686 352a 1001 695 551 689 312 936 32 1100 94 124 685 562 171 195a 8 (a)

source of isolation

IV

III

II

I

fermented apple fermented cabbage soured cream fermented cucumber cottage cheese fermented cow’s milk cottage cheese soured cream soured cream fermented cabbage fermented cabbage cottage cheese fermented cabbage fermented cabbage fermented cabbage fermented cabbage fermented cabbage fermented cabbage fermented cabbage fermented cabbage fermented cucumber bryndza fermented cabbage fermented cabbage fermented cabbage fermented cabbage fermented cabbage cottage cheese bryndza fermented cow’s milk bryndza type strain cottage cheese bryndza soured cream cottage cheese fermented cabbage soured cream fermented tomato fermented cabbage fermented cabbage fermented cabbage cottage cheese fermented cabbage cottage cheese fermented cabbage soured cream fermented cabbage fermented cabbage fermented cabbage fermented cabbage fermented cabbage fermented goat’s milk fermented cabbage fermented cabbage cottage cheese fermented cabbage cottage cheese fermented cabbage fermented cucumber fermented cabbage fermented cabbage fermented cabbage fermented cabbage fermented cabbage fermented cabbage bryndza bryndza bryndza soured cream soured cream fermented cabbage fermented cabbage fermented cabbage fermented cabbage fermented cabbage cottage cheese fermented cabbage cottage cheese fermented cabbage fermented cabbage bryndza cottage cheese fermented cabbage fermented cabbage cottage cheese cottage cheese cottage cheese cottage cheese cottage cheese cottage cheese soured cream fermented cabbage cottage cheese cottage cheese fermented cabbage cottage cheese cottage cheese cottage cheese fermented goat’s milk fermented cabbage soured cream fermented cabbage fermented cow’s milk soured cream cottage cheese cottage cheese soured cream bryndza soured cream

with pH 3

100%

with pH 2

90%

with pH 10

80%

with 4 % NaCl

Letters in Applied Microbiology 61, 283--292 © 2015 The Society for Applied Microbiology

70%

at 10°C at 45°C

Figure 1 (a) Dendrogram based on cellular fatty acids profiles of 110 Lactobacillus plantarum strains. Similarities were calculated as Pearson’s r product–moment correlation coefficient among cellular fatty acids profiles and clustering was performed using the UPGMA algorithm; (b) data of stress resistance profile for each strain obtained in our previous study (Vasyliuk et al. 2014b). Stressful culture conditions are listed at the top. ‘+’ growth observed under stress factor; ‘ ’ no growth observed under stress factor; ND: no determined.

60%

with 6 % NaCl with 8 % NaCl

50%

– + – + + – – – + + + + + – + + – + – – – – – – – + – – – – – – – + – – – – + – – – – + + – – – – + – + + – – – – + – – + – + – – + – + + + + – – + – – – + – – – + – + + + – – – + – – + – – – + + + + + – + – – + – + + + + + – + – + + + – – – + – – – + – – – + – + + + + + – + – – – – + – – + + + + + – – – + – – – – – – – + + + + – – – – + – + + – – – – + – + + + + – – + – – – – – – – + – + + + + + – – – – – + – – – + + + + + – – – + – + + + – – – + – + + + – – – + – – – – – – – + – + + – – – ND NDNDND ND NDNDND – + – + + + – – – + – + + – – – – + – – – + + – – + – – + – – – – + – – – – – – – + + + + – + – – + – + + – + – – + – + + + – – – + – + + – – – – + + + + – – – – + – + + – + – – + – – – – – – – + – – + – – – – + + + + – – – – + + + + – + + – + – – – + – – – + – + + – + – – + – + + + + – – + – + + + + – – – + + + + + – – + – + + – – – – + + + + + – – – + + + + – + + – + – + + – – – – + – – – + + – + + – – + – – – – + – + + + – – – + – + + – + – + + – + + + + + – + – + + + – – – + – + + – – – – + – + + + + – – + – + + – – – – + – + + – + + – + – + + – – – – + – + + – – – – + – + + – – – – + – – – + – – – – – – – – – – – – – + + – – – – + – – – – + – – + – + + + – – – + – + + + – – + + + + + – – – – + – + + + – – – + – + + – + + – + – + + + – – – + + + + – – – – + – + + + – – – + – + + – + – – + – + + – + – – + – – – – – – – + – + + – – – – + – + + – + – – + – + + – – – – + – + + – + – – + – – – – – – – + – – + – – – – – – – – + + – – – + + + – – – + + – + + + – – – + – + + + + – – + – – – – + – – + + + + – – – – + – + + – – – – + – + + – – – – + – + + – – – – + – – + + – – – + – + + – – – – – + + + – + – – + – + + + – – – – – – + – + – – – – + + + + – – + – + + – – – – + – + + – – – – + – – – – – – – + – + + – + – + – – + + + – – (b)

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CLUSTER I

CLUSTER II

Fermented cabbage 11%

Soured cream 10%

Bryndza 11%

Cottage cheese 22%

Fermented milk 3%

Fermented cabbage 42%

Soured cream 45%

Cottage cheese 42%

Fermented milk 11%

Bryndza 3% CLUSTER IV

CLUSTER III Fermented milk 3% Cottage cheese 13%

Soured cream 7%

Fermented apple 13% Fermented cucumber 13%

Bryndza 12%

Fermented cabbage 60%

Soured cream 25%

Fermented Fermented cucumber 3% tomato 2%

Fermented milk 12%

Fermented cabbage 12% Cottage cheese 25%

Figure 2 Relative proportions of Lactobacillus plantarum strains from different origins on each cluster.

Table 1 Fatty acids compositions of 110 Lactobacillus plantarum strains according to the cluster analysis results Fatty acids composition (%), mean  SD Fatty acids

Cluster I (82% strains)

C14:0 C16:0 C16:1 C18:0 cC18:1 tC18:1 cycC19:0

1535 35477 0582 13407 25426 3814 19754

      

2388a 10274b 0926c 5210 8407g 3685h 7732

Cluster II (30% strains) 1756 18273 1431 1656 14760 6664 55640

      

0926a 3988 0944cd 1330e 5649 3812 5130

Cluster III (546% strains) 1745 20791 1494 3290 27145 3730 41759

      

1610a 3839 1363d 3455f 3845g 2855h 7494j

Cluster IV (72% strains) 1407 32251 0660 1402 8778 12828 42648

      

0918a 4067b 0603cd 2072ef 4709 6431 4008j

Values in the same raw with the same letter are not significantly different (P > 005).

contained slightly greater levels of the C16:0 and tC18:1 fatty acids and lower levels of the cC18:1 than the strains obtained from fermented vegetables (Fig. 3a). It is well 286

known that environmental and physiological factors affect the fatty acids composition of bacteria (Gilarova et al. 1994). Generally, vegetables contain carbohydrates as the

Letters in Applied Microbiology 61, 283--292 © 2015 The Society for Applied Microbiology

Cellular fatty acids heterogeneity of Lact. plantarum strains

I. Garmasheva et al.

CLUSTER I

CLUSTER II

Fermented cabbage 11%

Soured cream 10%

Bryndza 11%

Cottage cheese 22%

Fermented milk 3%

Fermented cabbage 42%

Soured cream 45%

Cottage cheese 42%

Fermented milk 11%

Bryndza 3% CLUSTER IV

CLUSTER III Fermented milk 3% Cottage cheese 13%

Soured cream 7%

Fermented apple 13% Fermented cucumber 13%

Bryndza 12%

Fermented cabbage 60%

Soured cream 25%

Fermented Fermented cucumber 3% tomato 2%

Fermented milk 12%

Fermented cabbage 12% Cottage cheese 25%

Figure 2 Relative proportions of Lactobacillus plantarum strains from different origins on each cluster.

Table 1 Fatty acids compositions of 110 Lactobacillus plantarum strains according to the cluster analysis results Fatty acids composition (%), mean  SD Fatty acids

Cluster I (82% strains)

C14:0 C16:0 C16:1 C18:0 cC18:1 tC18:1 cycC19:0

1535 35477 0582 13407 25426 3814 19754

      

2388a 10274b 0926c 5210 8407g 3685h 7732

Cluster II (30% strains) 1756 18273 1431 1656 14760 6664 55640

      

0926a 3988 0944cd 1330e 5649 3812 5130

Cluster III (546% strains) 1745 20791 1494 3290 27145 3730 41759

      

1610a 3839 1363d 3455f 3845g 2855h 7494j

Cluster IV (72% strains) 1407 32251 0660 1402 8778 12828 42648

      

0918a 4067b 0603cd 2072ef 4709 6431 4008j

Values in the same raw with the same letter are not significantly different (P > 005).

contained slightly greater levels of the C16:0 and tC18:1 fatty acids and lower levels of the cC18:1 than the strains obtained from fermented vegetables (Fig. 3a). It is well 286

known that environmental and physiological factors affect the fatty acids composition of bacteria (Gilarova et al. 1994). Generally, vegetables contain carbohydrates as the

Letters in Applied Microbiology 61, 283--292 © 2015 The Society for Applied Microbiology

Cellular fatty acids heterogeneity of Lact. plantarum strains

I. Garmasheva et al.

agar, H. pylori strains showed 18:1 9c and increased 16:0 and 18:0 fatty acids content and decreased 14:0 content compared to strains grown on fatty acids-free agar. Differences in cellular fatty acids composition of Lact. plantarum strains could also be linked to the processing of the two types of raw materials (fresh vegetables and milk). Production of fermented vegetables requires no heating. In contrast, some dairy products, such as cottage cheese and bryndza, undergo heating during manufacturing. In addition, salt (up to 3%) is used in the bryndza processing. In our previous work, we have demonstrated that a relatively large variability in stress tolerance exists in Lact. plantarum strains, isolated from different fermented foods in Ukraine (Vasyliuk et al. 2014b). We have established the relationship between the ability to grow at stress conditions and source of isolation. Lactobacillus plantarum strains, isolated from fermented vegetables were more resistance to acid, alkaline and osmotic stress. Others authors have shown that plant-derived strains of LAB have some characteristics that differ from those of milk-derived strains (Gutierrez-Mendez et al. 2010). In addition, Fig. 1b shows data of stress resistance profile for each strain, obtained in our previous study (Vasyliuk et al. 2014b). Strains of each cluster are quite heterogeneous in their ability to grow under stress conditions, except for strains of clusters I and IV that did not grow at pH 2 and pH 10 respectively. Multivariate analysis for the correlation between cellular fatty acids contents and ability to grow at different stress factors was performed for strains depending on the cluster and the source from which strains were isolated (Table 2). There were significant strong positive correlations between relative content of C14:0 and growth at 45°C (r = 085), strong negative correlations between content tC18:1 and growth at pH 10 (r = 077) among strains of cluster I and between content C18:0 and growth at 10°C among strains of cluster IV (P < 005). In clusters II and III there were a weak negative correlations between ability to growth at 10°C and amounts of C16:1 (r = 037) and cC18:1 (r = 031) respectively. No correlation between fatty acids composition of vegetable-derived Lact. plantarum strains and stress resistance was detected. By contrast, in dairy-derived strains, we observed a weak negative correlations (r = 036) between growth at 10°C and levels of hexadecanoic acid, between growth at 45°C and levels of and cyclopropane fatty acid (r = 030). Weak positive correlations were obtained between growth at 45°C and levels of C14:0 (r = 040), cC18:1 (r = 030) and C18:0 (r = 033) acids. In addition, there was strong negative correlation between content tC18:1 and growth at pH 10 (r = 089) and significant strong positive correlations between relative 288

content of C14:0 and growth at 10°C (r = 089) and between content of cycC19:0 and growth at pH 10 among strains isolated from fermented milk. A strong positive correlation was obtained between growth at 45°C (r = 079) and levels of C14:0 among strains isolated from soured cream. In addition, we observed a strong negative correlation between relative content of tC18:1 and growth at pH 3 (r = 071) among bryndza-derived strains. In contrast, no correlation between fatty acids composition of cottage cheese-derived Lact. plantarum strains and their stress resistance was detected. Also, we carried out the analysis of the correlation between the source isolation and composition of cellular fatty acids. Amounts of C16:0, tC18:1 and cC18:1 were correlated with type of fermented foods – fermented vegetables or dairy products. Analysis of the correlation between the type of dairy products from which strains were isolated and fatty acids profiles showed a weak correlations between cottage cheese and cC18:1 and tC18:1, between bryndza and C14:0, and between soured cream and C16:0 and cycC19:0 (Table 2). So, all fatty acids detected in this work have been identified previously in cellular membrane of lactic acid bacteria by others authors (Suutari and Laakso 1992; Dykes et al. 1995). It is known that oleic acid is incorporated into LAB cellular membranes if bacteria are grown on the medium supplemented with Tween 80 (Polacheck et al. 1966). It was also shown for the majority of LAB strains that the octadecenoic acid in a cell membrane can be converted into the corresponding cyclopropane fatty acids (Polacheck et al. 1966; Smith and Norton 1980). Cyclopropane fatty acids are regarded by many authors as the major fatty acids in LAB (Suutari and Laakso 1992; Dionisi et al. 1999) and they may play a role in bacterial adaptation to environmental stresses. In fact, authors (G omez Zavaglia et al. 2000) reported that these cyclopropane fatty acids enhanced the stress tolerance of L. delbrueckii subsp. bulgaricus, L. helveticus and L. acidophilus, as well as that the amount of cyclopropane fatty acids in the membranes of numerous LAB increased under various stress situations (Guillot et al. 2000; Beal et al. 2001). The crucial role of unsaturated fatty acids has been reported by several works and in response to several different stresses, including low or high growth temperatures, oxidative stress, acid stress and salt addition stress (Streit et al. 2008; Montanari et al. 2010; Wu et al. 2012). Currently, it is known that relative content of fatty acids can be varied under the influence of stress factors during cultivation. At high temperature, the level of C16:0 decreases while C18:1 and cycC19:0 levels rise as was shown for Lactococcus lactis strain (Guillot et al. 2000). Lower cultivation temperature (compared with the optimum) resulted in lower production of lactobacillic

Letters in Applied Microbiology 61, 283--292 © 2015 The Society for Applied Microbiology

Cellular fatty acids heterogeneity of Lact. plantarum strains

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Table 2 Correlation coefficients between cellular fatty acids composition and ability to grow under stress conditions and origin of Lactobacillus plantarum strains Fatty acids Stress factors/origin Strains of Cluster I* pH 2–3 pH 10 NaCl 4–8% 10°C 45°C Strains of Cluster II pH 2–3 pH 10 NaCl 4–8% 10°C 45°C Strains of Cluster III pH 2–3 pH 10 NaCl 4–8% 10°C 45°C Strains of Cluster IV pH 2–3 pH 10 NaCl 4–8% 10°C 45°C Strains isolated from pH 2–3 pH 10 NaCl 4–8% 10°C 45°C Strains isolated from pH 2–3 pH 10 NaCl 4–8% 10°C 45°C Strains isolated from pH 2–3 pH 10 NaCl 4–8% 10°C 45°C Strains isolated from pH 2–3 Alkali NaCl resistance 10°C 45°C Strains isolated from pH 2–3 Alkali NaCl resistance

C14:0

fermented vegetables

dairy products

fermented milk

cottage cheese

bryndza

C16:0

C16:1

040 025 025 008 085‡

012 021 054 022 0.37

026 047 060 060 023

001 008 007 008 016

004 017 006 0.12 011

021 012 012 012 001 059 –† 010 017 019 010 012 001 013 005 001 010 017 005 004 005 040‡ 001 077 018 041 089‡ – 014 006 009 008 001 016 022‡ 037 015 –

cC18:1

tC18:1

C18:0

cycC19:0

003 002 010 004 060

011 078‡ 054 047 039

064 006 013 041 008

044 005 012 022 026

024 012 026 037‡ 021

009 002 008 008 002

007 014 001 011 005

007 003 017 015 010

009 012 016 021 002

010 023 009 004 001

001 018 014 016 001

001 024 008 031‡ 005

010 007 007 001 009

015 019 005 016 022

001 021 001 017 010

021 – 057 031 039 020‡ 007 005 001 003 009 020‡ 001 006 010 036‡ 003 013 030 001 004 080 – 001 018 011 009 010 006 005 049 010 –

034 – 001 009 025 010 005 015 013 003 003 010 020 011 017 019 012 004 061 026 004 074 – 003 027 030 014 018 005 009 010 006 –

047 – 057 026 031 020‡ 014 008 003 016 023 020‡ 003 007 017 008 030‡ 009 009 037 013 050 – 028‡ 031 002 009 002 027 015 005 009 –

027 – 020 010 001 030‡ 001 018 003 002 015 030‡ 007 017 010 009 009 011 007 089‡ 050 054 – 028‡ 011 013 007 024 005 002 060 028 –

024 – 033 094‡ 005 010 010 026 005 007 005 010 001 003 008 026 033‡ 007 088 018 006 088 – 002 027 013 008 003 023 006 071‡ 012 –

004 – 056 030 001 010 017 004 004 012 009 010 003 002 017 022 030‡ 009 059 092‡ 005 049 – 014 033 011 011 002 026 015 014 005 – (Continued)

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agar, H. pylori strains showed 18:1 9c and increased 16:0 and 18:0 fatty acids content and decreased 14:0 content compared to strains grown on fatty acids-free agar. Differences in cellular fatty acids composition of Lact. plantarum strains could also be linked to the processing of the two types of raw materials (fresh vegetables and milk). Production of fermented vegetables requires no heating. In contrast, some dairy products, such as cottage cheese and bryndza, undergo heating during manufacturing. In addition, salt (up to 3%) is used in the bryndza processing. In our previous work, we have demonstrated that a relatively large variability in stress tolerance exists in Lact. plantarum strains, isolated from different fermented foods in Ukraine (Vasyliuk et al. 2014b). We have established the relationship between the ability to grow at stress conditions and source of isolation. Lactobacillus plantarum strains, isolated from fermented vegetables were more resistance to acid, alkaline and osmotic stress. Others authors have shown that plant-derived strains of LAB have some characteristics that differ from those of milk-derived strains (Gutierrez-Mendez et al. 2010). In addition, Fig. 1b shows data of stress resistance profile for each strain, obtained in our previous study (Vasyliuk et al. 2014b). Strains of each cluster are quite heterogeneous in their ability to grow under stress conditions, except for strains of clusters I and IV that did not grow at pH 2 and pH 10 respectively. Multivariate analysis for the correlation between cellular fatty acids contents and ability to grow at different stress factors was performed for strains depending on the cluster and the source from which strains were isolated (Table 2). There were significant strong positive correlations between relative content of C14:0 and growth at 45°C (r = 085), strong negative correlations between content tC18:1 and growth at pH 10 (r = 077) among strains of cluster I and between content C18:0 and growth at 10°C among strains of cluster IV (P < 005). In clusters II and III there were a weak negative correlations between ability to growth at 10°C and amounts of C16:1 (r = 037) and cC18:1 (r = 031) respectively. No correlation between fatty acids composition of vegetable-derived Lact. plantarum strains and stress resistance was detected. By contrast, in dairy-derived strains, we observed a weak negative correlations (r = 036) between growth at 10°C and levels of hexadecanoic acid, between growth at 45°C and levels of and cyclopropane fatty acid (r = 030). Weak positive correlations were obtained between growth at 45°C and levels of C14:0 (r = 040), cC18:1 (r = 030) and C18:0 (r = 033) acids. In addition, there was strong negative correlation between content tC18:1 and growth at pH 10 (r = 089) and significant strong positive correlations between relative 288

content of C14:0 and growth at 10°C (r = 089) and between content of cycC19:0 and growth at pH 10 among strains isolated from fermented milk. A strong positive correlation was obtained between growth at 45°C (r = 079) and levels of C14:0 among strains isolated from soured cream. In addition, we observed a strong negative correlation between relative content of tC18:1 and growth at pH 3 (r = 071) among bryndza-derived strains. In contrast, no correlation between fatty acids composition of cottage cheese-derived Lact. plantarum strains and their stress resistance was detected. Also, we carried out the analysis of the correlation between the source isolation and composition of cellular fatty acids. Amounts of C16:0, tC18:1 and cC18:1 were correlated with type of fermented foods – fermented vegetables or dairy products. Analysis of the correlation between the type of dairy products from which strains were isolated and fatty acids profiles showed a weak correlations between cottage cheese and cC18:1 and tC18:1, between bryndza and C14:0, and between soured cream and C16:0 and cycC19:0 (Table 2). So, all fatty acids detected in this work have been identified previously in cellular membrane of lactic acid bacteria by others authors (Suutari and Laakso 1992; Dykes et al. 1995). It is known that oleic acid is incorporated into LAB cellular membranes if bacteria are grown on the medium supplemented with Tween 80 (Polacheck et al. 1966). It was also shown for the majority of LAB strains that the octadecenoic acid in a cell membrane can be converted into the corresponding cyclopropane fatty acids (Polacheck et al. 1966; Smith and Norton 1980). Cyclopropane fatty acids are regarded by many authors as the major fatty acids in LAB (Suutari and Laakso 1992; Dionisi et al. 1999) and they may play a role in bacterial adaptation to environmental stresses. In fact, authors (G omez Zavaglia et al. 2000) reported that these cyclopropane fatty acids enhanced the stress tolerance of L. delbrueckii subsp. bulgaricus, L. helveticus and L. acidophilus, as well as that the amount of cyclopropane fatty acids in the membranes of numerous LAB increased under various stress situations (Guillot et al. 2000; Beal et al. 2001). The crucial role of unsaturated fatty acids has been reported by several works and in response to several different stresses, including low or high growth temperatures, oxidative stress, acid stress and salt addition stress (Streit et al. 2008; Montanari et al. 2010; Wu et al. 2012). Currently, it is known that relative content of fatty acids can be varied under the influence of stress factors during cultivation. At high temperature, the level of C16:0 decreases while C18:1 and cycC19:0 levels rise as was shown for Lactococcus lactis strain (Guillot et al. 2000). Lower cultivation temperature (compared with the optimum) resulted in lower production of lactobacillic

Letters in Applied Microbiology 61, 283--292 © 2015 The Society for Applied Microbiology

Cellular fatty acids heterogeneity of Lact. plantarum strains

I. Garmasheva et al.

Table 2 Correlation coefficients between cellular fatty acids composition and ability to grow under stress conditions and origin of Lactobacillus plantarum strains Fatty acids Stress factors/origin Strains of Cluster I* pH 2–3 pH 10 NaCl 4–8% 10°C 45°C Strains of Cluster II pH 2–3 pH 10 NaCl 4–8% 10°C 45°C Strains of Cluster III pH 2–3 pH 10 NaCl 4–8% 10°C 45°C Strains of Cluster IV pH 2–3 pH 10 NaCl 4–8% 10°C 45°C Strains isolated from pH 2–3 pH 10 NaCl 4–8% 10°C 45°C Strains isolated from pH 2–3 pH 10 NaCl 4–8% 10°C 45°C Strains isolated from pH 2–3 pH 10 NaCl 4–8% 10°C 45°C Strains isolated from pH 2–3 Alkali NaCl resistance 10°C 45°C Strains isolated from pH 2–3 Alkali NaCl resistance

C14:0

fermented vegetables

dairy products

fermented milk

cottage cheese

bryndza

C16:0

C16:1

040 025 025 008 085‡

012 021 054 022 0.37

026 047 060 060 023

001 008 007 008 016

004 017 006 0.12 011

021 012 012 012 001 059 –† 010 017 019 010 012 001 013 005 001 010 017 005 004 005 040‡ 001 077 018 041 089‡ – 014 006 009 008 001 016 022‡ 037 015 –

cC18:1

tC18:1

C18:0

cycC19:0

003 002 010 004 060

011 078‡ 054 047 039

064 006 013 041 008

044 005 012 022 026

024 012 026 037‡ 021

009 002 008 008 002

007 014 001 011 005

007 003 017 015 010

009 012 016 021 002

010 023 009 004 001

001 018 014 016 001

001 024 008 031‡ 005

010 007 007 001 009

015 019 005 016 022

001 021 001 017 010

021 – 057 031 039 020‡ 007 005 001 003 009 020‡ 001 006 010 036‡ 003 013 030 001 004 080 – 001 018 011 009 010 006 005 049 010 –

034 – 001 009 025 010 005 015 013 003 003 010 020 011 017 019 012 004 061 026 004 074 – 003 027 030 014 018 005 009 010 006 –

047 – 057 026 031 020‡ 014 008 003 016 023 020‡ 003 007 017 008 030‡ 009 009 037 013 050 – 028‡ 031 002 009 002 027 015 005 009 –

027 – 020 010 001 030‡ 001 018 003 002 015 030‡ 007 017 010 009 009 011 007 089‡ 050 054 – 028‡ 011 013 007 024 005 002 060 028 –

024 – 033 094‡ 005 010 010 026 005 007 005 010 001 003 008 026 033‡ 007 088 018 006 088 – 002 027 013 008 003 023 006 071‡ 012 –

004 – 056 030 001 010 017 004 004 012 009 010 003 002 017 022 030‡ 009 059 092‡ 005 049 – 014 033 011 011 002 026 015 014 005 – (Continued)

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plantarum genome diversity by using microarrays. J Bacteriol 187, 6119–6127. Montanari, C., Sado Kamdem, S.L., Serrazanetti, D.I., Etoa, F. and Guerzoni, M.E. (2010) Synthesis of cyclopropane fattyacids in Lactobacillus helveticus and Lactobacillus sanfranciscensis and their cellular fatty acids changes following short term acid and cold stresses. Food Microbiol 27, 493–502. Polacheck, J.W., Tropp, B.E., Law, J.H. and McCloskey, J.A. (1966) Biosynthesis of cyclopropane compounds. J Biol Chem 241, 3362–3364. Scherer, C., M€ uller, K.-D., Rath, P.-M. and Ansorg, R.A.M. (2003) Influence of culture conditions on the fatty acid profiles of laboratory-adapted and freshly isolated strains of Helicobacter pylori. J Clin Microbiol 41, 1114–1117. Siezen, R.J., Tzeneva, V.A., Castioni, A., Wels, M., Phan, H.T., Rademaker, J.L., Starrenburg, M.J., Kleerebezem, M. et al. (2010) Phenotypic and genomic diversity of Lactobacillus plantarum strains isolated from various environmental niches. Environ Microbiol 12, 758–773. Smith, D.D. Jr and Norton, S.J. (1980) S-Adenosylmethionine, cyclopropane fatty acid synthase, and the production of lactobacillic acid in Lactobacillus plantarum. Arch Biochem Biophys 205, 564–570. Streit, F., Delettre, J., Corrieu, G. and Beal, C. (2008) Acid adaption of Lactobacillus delbrueckii subsp. bulgaricus induces physiological responses at membrane and cytosolic levels that improves cryotolerance. J Appl Microbiol 105, 1071–1080. Suutari, M. and Laakso, S. (1992) Temperature adaptation in Lactobacillus fermentum: interconversions of oleic, vaccenic

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and dihydrosterculic acids. J Gen Microbiol 138, 445–450. Tanganurat, W., Quinquis, B., Leelawatcharamas, V. and Bolotin, A. (2009) Genotypic and phenotypic characterization of Lactobacillus plantarum strains isolated from Thai fermented fruits and vegetables. J Basic Microbiol 49, 377–385. Turchi, B., Mancini, S., Fratini, F., Pedonese, F., Nuvoloni, R., Bertelloni, F., Ebani, V.V. and Cerri, D. (2013) Preliminary evaluation of probiotic potential of Lactobacillus plantarum strains isolated from Italian food products. World J Microbiol Biotechnol 29, 1913–1922. Vasyliuk, O.M., Kovalenko, N.K., Harmasheva, I.L. and Oleshchenko, L.T. (2014a) Isolation and identification of bacteria of Lactobacillus genus from fermented products in different regions of Ukraine. Mikrobiol Z 2, 2–9. Vasyliuk, O.M., Kovalenko, N.K. and Harmasheva, I.L. (2014b) Physiological and biochemical properties of the Lactobacillus plantarum, isolated from traditional fermented products of Ukraine. Mikrobiol Z 5, 2–8. Wu, C., Zhang, J., Wang, M., Du, G. and Chen, J. (2012) Lactobacillus casei combats acid stress by maintaining cell membrane functionality. J Ind Microbiol Biotechnol 39, 1031–1039. Xanthopoulos, V., Hatzikamari, M., Adamidis, T., Tsakalidou, E., Tzanetakis, N. and Litopoulou-Tzanetaki, E. (2000) Heterogeneity of Lactobacillus plantarum isolates from feta cheese throughout ripening. J Appl Microbiol 88, 1056–1064.

Letters in Applied Microbiology 61, 283--292 © 2015 The Society for Applied Microbiology

Intraspecies cellular fatty acids heterogeneity of Lactobacillus plantarum strains isolated from fermented foods in Ukraine.

The intraspecies heterogeneity of cellular fatty acids composition of Lactobacillus plantarum strains isolated from Ukrainian traditional fermented fo...
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